The present invention relates generally to cryogenic preservation and more particularly to a method of preserving for examination and diagnostic purposes.
Biological materials such as tissues are subjected to various treatments in an histology laboratory to prepare specimens on slides for viewing under a microscope. Pathologists carefully examine the slides and report their findings, which aids physicians in the diagnosis of disease or disease processes. Histopathology has traditionally relied upon examination of samples prepared by one of two basic methods. In the first histological method, samples undergo significant processing in the laboratory, such as fixation to preserve tissues, dehydration to remove water from tissues, infiltration with embedding agents such as paraffin, embedment, sectioning or cutting sections of the tissue for placement on a slide, mounting the sections, and staining the sections to enhance details. The second method, cryogenic preparation, significantly reduces the processing of the first method in that it generally involves snap freezing in a cold liquid or environment, sectioning, mounting, and staining.
While the first method yields significantly superior visualization, it requires an extended period of time for processing, generally a minimum of 18 to 24 hours. Thus this method cannot be applied in situations where a rapid diagnosis of a pathologic process is required, such as during a surgical procedure. Additionally, the processing techniques employed may destroy all or part of the biological activity of the tissues.
The second method has the advantage of speed (30 minutes to 1 hour), however tissue specimens prepared using cryogenic preparation are often subject to cellular damage due to ice crystal formation, which can also cause the loss of biological function of molecules of interest within the tissues, and overall loss of tissue integrity manifested as degraded anatomical structure. Many commercial pathology laboratories discourage the use of frozen tissue for immunohistochemistry in all but special circumstances, because ice crystal formation in stored tissue causes many abnormal artifacts within the sample which make diagnostic interpretation quite difficult, or even impossible in some cases.
With the advent of poly- and then monoclonal antibodies, the focus of both traditional microscopic histology and pathology has shifted from simple subjective observation, to direct objective staining procedures. These newer immunohistochemistry (IHC) techniques help in determining diagnosis when histopathology alone proves inconclusive. However, IHC techniques are dependent on biologically intact receptors within the specimen for proper staining to occur. Therefore it is desirable to utilize a method of tissue specimen preparation that does not limit the amount of active biological material present after preparation is complete.
Therefore, what is needed is an improved way to cryogenically preserve viable single cells, tissues, organs, nucleic acids, or other biologically active molecules, that avoids at least some of the problems inherent in currently available methods. Accordingly, the present invention provides a method of cryopreservation for freezing a biochemically active tissue sample by immersing the sample in cooling fluid and circulating the cooling fluid past the material. The cooling fluid is circulated past the tissue sample at a substantially constant, predetermined velocity and temperature to freeze the tissue sample such that it is vitrified, yet the tissue sample maintains its anatomical structure and remains biochemically active after thaw. In at least one embodiment, the cooling fluid is maintained at a temperature of between about xe2x88x9220 degrees centigrade and xe2x88x9230 degrees centigrade, and the velocity of the cooling fluid past the tissue sample is about 35 liters per minute per foot of cooling fluid through an area not greater than about 24 inches wide and 48 inches deep. Additionally, at least one embodiment of the present invention immerses a biologically active tissue sample in cooling fluid to freeze the sample directly to a temperature higher than about xe2x88x9230 degrees centigrade. A further embodiment of the present invention provides for circulating the cooling fluid past a multi-path heat exchanging coil submersed in the cooling fluid, where the heat exchanging coil is capable of removing at least the same amount of heat from the cooling fluid as the cooling fluid removes from the tissue sample. At least one embodiment provides a system for implementing the above mentioned methods.
An object of at least one embodiment of the present invention is application of a method to freeze biological material wherein the formation of ice crystals and stress fractures is avoided, and cellular biochemical function is maintained after freezing.
An advantage of at least one embodiment of the present invention is that cryopreservation recovery rates are significantly increased, because biological material is vitrified during freezing.
Another advantage of at least one embodiment of the present invention is that cryopreservation recovery rates are improved, because biological material is vitrified at a high enough temperature to avoid the formation of stress fractures within cell membranes.
Another advantage of at least one embodiment of the present invention is that cryopreservation recovery rates are such that a considerably higher percentage of the biological material maintains its anatomical structure and remains biochemically active after thaw as compared to currently available methods.
An additional advantage of at least one embodiment of the present invention is that cryopreservation recovery rates are such that the biological material samples lend themselves to the application of sectioning, processing and subsequent histological, ultrastructural, and immunohistochemistry examination in shorter periods of time than traditional pathology techniques, thus shortening time to results.
A further advantage of at least one embodiment of the present invention is that once frozen, existing cryopreservation storage facilities and mechanisms can be used to store the frozen biological materials.